Saturday, December 26, 2015

A Gentle Touch at Metaphase

Dynamic modeling of the metaphase chromosomes as microtubules push them about.

One of the more magical phenomena of biology is the orchestrated congregation and division of chromosomes at the midline of a eukaryotic cell at mitosis, or cell division. One has to keep reminding oneself that there is no central brain organizing the process- it is driven by a network of molecules regulating each other and conjuring collective organization and action out of blind chemistry.

What are the mechanics involved? The main force comes from the microtubules originating at the spindle poles, which connect in somewhat noisy fashion to the chromosomes, especially to the kinetochores that are located at the middle of each chromosome at its centromere. About 10 to 30 microtubule plus ends are stuck into each kinetochore, when everything is working properly. Microtubules are fascinating structures in themselves- they can exert force in either direction, either (+) growing by polymerizing more of their subunits, or (-) by shrinking and losing subunits. This is quite apart from the various cargo carriers that use the motor kinesin to travel along stable microtubules to ferry materials around cells.

Electron microscopy of microtubule ends (bottom), and as stuck into
a kinetochore (at right). When splayed out, the end is de-polymerizing
and retreating. When straight, the end is advancing by polymerization.

The strongest forces come from the microtubules docked successfully to kinetochores. Having all the kinetochores docked is a key prerequisite for proceeding with cell division, to insure that each daughter cell gets a full set of chromosome copies. But there is another, weaker force, which comes from microtubules touching other areas of the chromosomes, away from the kinetochores. These attachments (called the polar ejection force, or PEF) are much simpler, and only push in one direction, away from the spindle and towards the midline of the cell. They connect to chromokinesins distributed all over the chromosome arms. A recent paper modeled what is known about these forces, and concluded that it is the PEF that gives the key nudge to line all the chromosomes up properly at the midline, while the kinetochore-attached forces swing rather wildly back and forth, causing the chromosomes to oscillate as they gradually find their equilibrium position.

A still from a video that shows the typical oscillation
of chromosomes being tugged back and forth by
their attached microtubules. The chromosomes are
the dark globules beneath the labelled molecules (red
is the microtubules, green is the kinetochores and
spindle poles).

A third force or factor is the attachment strength between the two copied chromosomes, the two partnered kinetochores. This is trivial, however, as it acts like a rather stiff spring which can not be broken until the signal is given for the cell actually divide and the paired chromosomes part to opposite sides.

The researchers use video recordings of labelled human HeLa cells to document the back-and-forthings of the mitotic chromosomes, and using a basic model of the forces involved develop a more detailed model of the dynamics they are seeing. The upshot is that while the strong kinetochore forces are switching back and forth, (and forms another story about how the kinetochore-microtubule connections are made and regulated), insuring capture of each kinetochore, the PEF, which is about 1/3 as strong, consistently pushes each chromosome towards the center, thus biassing the net force to line everyone up at the midline. These two processes are collaborative, since alignment in a single row / plate at the center also helps to insure that all the kinetochores are captured, on both sides. There is an additional consideration, which is that each kinetochore needs to associate with only one spindle, not with both. This is probably helped by the stochastic push-pull process, where microtubules, which are relatively stiff themselves, rapidly attach and detach from the kinetochore, which has a rigid, single orientation.

Schema and results from the cited paper. On left are the forces at work,
including a spring force between the paired kinetochores (green). The other
graphs show the deduced forces for individual kinetochore excursions,
with the PEF and inter-kinetochore spring force (right) much weaker than
the main kinetochore microtubules, which show much more dramatic
directional switching.

Once each kinetochore has successfully docked with microtubules from the opposite spindle pole, how are their motions coordinated? The researchers find that switches in direction are led 4/5 of the time by the side that is shorter and pulling towards its pole. The stiff connection between the two chromosomes then quickly transmits this switch in motion to induce the partner kinetochore+microtubules to follow suit, after which the pair is once again heading together in the opposite, direction. The bias in the tendency to switch directions also helps keep the chromosomes centrally postioned. But why does this bias happen? That remains unanswered, and likely is also due to the gentle, but persistent, PEF.

All this is a prelude, once the spindle checkpoint is passed and the paired chromosomes fire their release rockets, to when the kinetochore-associated microtubules pull hard and in unison toward the poles. This transition orchestrated by other molecular signals which have been well-studied, particularly the anaphase promoting complex. Life is a process, and a network, which knows no end and few boundaries.

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