Contents - Max-Planck-Institut für Physik komplexer Systeme

2.28 Kinetochores are Captured by Microtubules Performing Random Angular Movement
IANA KALININA, AMITABHA NANDI, ALEXANDER KRULL, BENJAMIN LINDNER,
NENAD PAVIN, IVA M.TOLIĆ-NØRRELYKKE
In living cells, proper segregation of genetic material
between the two daughter cells requires all chromosomes
to be connected to the spindle microtubules
(MTs). Linkers between chromosomes and MTs are
kinetochores (KCs), protein complexes on the chromosome.
In fission yeast, KCs are at the spindle pole
body (SPB), which facilitates their interaction with
MTs that grow from the SPB. If the spindle is disassembled,
it is able to recover including capturing KCs
that have been lost in the nucleoplasm. It is, however,
unknown how MTs find lost KCs. We found
that lost KCs can be captured by MTs performing
random angular movement. By using live cell imaging,
we observed that astral MTs pivot around the
SPB, in cell with and without lost KCs. By studying
the relationship between the MT angular movement
and MT length, we found that this movement is
most likely driven by thermal fluctuations. In addition,
we found that KCs and astral MTs by performing
random movement explore a comparable fraction
of space. Finally, by introducing a theoretical model,
we show that the process of KC capture can be explained
by the observed random movement of astral
MTs and of the KC.
A pioneering idea that could explain how MTs find
KCs is based on MT dynamics [1]. In this search-andcapture
scenario, the MTs are assumed to grow in random
directions from the centrosomes. If a MT does
not interact with a KC, it will undergo catastrophe and
shrink back to the centrosome. New MTs are nucleated,
each of them having a chance to reach a KC. At some
point, a lucky MT will capture a KC and became stabilized
by this interaction. Eventually, all KCs will get
captured. This mechanism, however, relies on a large
number of MTs and their high dynamicity. We hypothesize
that a so-far unknown mechanism may exist to
increase the efficiency of KC capture.
To study how MTs search for KCs we use fission yeast,
where we can follow the dynamics of each astral MT
during its lifetime, due to a small number of MTs. At
the onset of mitosis in fission yeast, KCs are close to the
duplicated spindle pole body (SPB). The SPBs nucleate
MTs that form a spindle and attach to KCs (Fig. 1). To
detach KCs from MTs, we use the lost kinetochore assay,
where MTs are disassembled by exposing the cells
in early mitosis to cold stress [2]. This type of stress
most likely also occurs in nature. After MT disassembly,
a large fraction of KCs are lost in the nucleoplasm,
while the remaining KCs are at the SPB. Once the stress
is relieved, MTs regrow from the SPB. The MTs that
grow in the direction of the other SPB contact the MTs
growing from that SPB, thereby reassembling the spindle
(spindle MTs), while several other MTs grow in
other directions (astral MTs). Astral MTs capture lost
KCs, retrieve it to the SPB, and mitosis progresses regularly
[2] (Fig. 1).
A
B
C
D
G
Figure 1: Mitosis in fission yeast. (A) At the onset of mitosis,
the spindle pole bodies (small grey spheres) nucleate microtubules
(green) that form a spindle and attach to kinetochores (red). (B) In
metaphase, kinetochores oscillate on the spindle. (C) In anaphase
A, sister kinetochores separate from each other and move to opposite
spindle poles. (D) If microtubules depolymerize during
prometaphase, kinetochores may get lost in the nucleoplasm. (E)
When microtubules re-polymerize, spindle re-forms. (F) An astral
microtubule captures the lost kinetochore. (G) The captured kinetochore
is retrieved to the spindle pole body, and (B) subsequently
moves on the spindle. Thus, a normal metaphase is established
again.
We imaged cells with KCs labeled in red and MTs in
green and found that after 1 hour of cold treatment
(4 ◦ C), MTs were absent and 60% of cells in metaphase
had one or more lost KCs. When the cells were rewarmed
to 25 ◦ C, the number of cells with lost KCs was
halved within 4 minutes. Live cell imaging revealed
that astral MTs, which are straight, change their orientation
with respect to the cell and to the spindle, where
one end of the MT is attached to the SPB and the other
end moves in the nucleoplasm (Fig. 2). This pivoting of
the MTs around the SPB changes the distance between
the MTs and the lost KC. The distance between one of
the MTs and the lost KC eventually diminishes and this
MT captures the lost KC (Fig. 2). The attachment of the
KC is typically close to the MT tip but attachment away
from the tip was also observed. These findings suggest
that pivoting of astral MTs may play a crucial role in
finding lost KCs.
96 Selection of Research Results
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